Y. Tanaka performed the technical improvement of Y2H. This work is supported by JSPS KAKENHI Grant Number JP22K06336 and the Institute for Fermentation, Osaka.

1. Construction of the mutant library
<ol>
	<li>Set up the polymerase chain reaction (PCR) (an example is shown below). Generally, prepare 50 &#181;L of the reaction, which is divided into ten aliquots of 5 &#181;L, and amplify the target gene fragment in a PCR machine4. 
	NOTE: Users should select an appropriate polymerase to efficiently introduce mutations. If the DNA fragment to be amplified is short, then a polymerase with a higher error rate, such as Taq polymerase, wh...

Biomolecular Interaction Studies Using Yeast Two-Hybrid Technique 

<ol>
	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Leanna, C. A., Hannink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reverse+two-hybrid+system:+A+genetic+scheme+for+selection+against+specific+protein/protein+interactions.">The reverse two-hybrid system: A genetic scheme for selection against specific protein/protein interactions.</a> Nucleic Acids Res. 24 (17), 3341-3347 (1996).</li><li>Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+two-hybrid+and+one-hybrid+systems+to+detect+dissociation+of+protein-protein+and+DNA-protein+interactions.">Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.</a> Proc Natl Acad Sci U S A. 93 (19), 10315-10320 (1996).</li><li>Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+isolation+of+interaction-null/impaired+mutants+using+the+yeast+two-hybrid+technique.">An efficient method for the isolation of interaction-null/impaired mutants using the yeast two-hybrid technique.</a> Genes Cells. 24 (12), 781-788 (2019).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. 4th edn. , (2012).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Birnboim, H. C., Doly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+alkaline+extraction+procedure+for+screening+recombinant+plasmid+DNA.">A rapid alkaline extraction procedure for screening recombinant plasmid DNA.</a> Nucleic Acids Res. 7 (6), 1513-1523 (1979).</li><li>Bartel, P. L., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein-protein+interactions+using+two-hybrid+system.">Analyzing protein-protein interactions using two-hybrid system.</a> Meth Enzymol. 254, 241-263 (1995).</li><li>Hollenberg, S. M., Sternglanz, R., Cheng, P. F., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol Cell Biol. 15 (7), 3813-3822 (1995).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Meth Enzymol. 350, 87-96 (2002).</li><li>Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+protein+extraction+from+yeast.">Rapid and reliable protein extraction from yeast.</a> Yeast. 16 (9), 857-860 (2000).</li><li>Van Deursen, F., Sengupta, S., De Piccoli, G., Sanchez-Diaz, A., Labib, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+associates+with+the+loaded+DNA+helicase+at+replication+origins+and+defines+a+novel+step+in+its+activation.">Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.</a> EMBO J. 31 (9), 2195-2206 (2012).</li><li>Watase, G., Takisawa, H., Kanemaki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+plays+a+role+in+functioning+of+the+eukaryotic+replicative+DNA+helicase,+cdc45-mcm-gins.">Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, cdc45-mcm-gins.</a> Current Biology: CB. 22 (4), 343-349 (2012).</li><li>Miyazawa-Onami, M., Araki, H., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-initiation+complex+assembly+functions+as+a+molecular+switch+that+splits+the+mcm2-7+double+hexamer.">Pre-initiation complex assembly functions as a molecular switch that splits the mcm2-7 double hexamer.</a> EMBO Rep. 18 (10), 1752-1761 (2017).</li></ol>

The authors declare that they have no conflict of interest.

This article describes how to isolate interaction-null/impaired mutants using the Y2H assay. Such mutants are powerful tools to analyze the function of a protein of interest. To isolate such mutants, rY2H assays were developed previously by modifying the Y2H host strain2,3. However, they have not greatly reduced the amount of labor. By contrast, mutants can be isolated with this method without a significant amount of labor. In this method, modification of the Y2H...

Interactions between biomolecules are the most basic part of biological phenomena. Protein-protein interactions constitute a significant part of such interactions. Therefore, identification of the interaction partner(s) of a protein of interest is critical to further elucidate the function of the protein/gene of interest. The yeast two-hybrid (Y2H) method is a popular technique to identify protein-protein interactions in vivo1. In this system, two proteins (X and Y) whose interaction is to be tested are fused to the DNA-binding (DB) domain and transcriptional activation domain (AD), respectively. The DB-X fusion protein binds to a recognition sequence of the DB domain; therefore, when proteins X and Y interact, the AD-Y fusion protein comes into the proximity of the recognition sequence. Consequently, transcription of the reporter gene downstream of the recognition sequence is activated. Therefore, the presence or absence of reporter gene activity can be used to determine the presence or absence of the protein-protein interaction1.
Once a specific interaction partner of the protein of interest is identified, further analyses should be performed to elucidate the biological function of the interaction. For this purpose, if mutants of the proteins that impair or remove the specific protein-protein interaction can be isolated, they will serve as powerful tools. The Y2H system can be used directly to isolate such mutants by screening 'interaction-negative' clones, starting with the wild-type 'interaction-positive' clone. To accelerate this process, 'reverse' Y2H (rY2H) systems were developed2,3. In rY2H systems, the host yeast strains harbor counter-selectable marker genes as reporter genes, meaning yeast cells grow only when the AD-Y and DB-X proteins do not interact.
Although both the Y2H and rY2H systems allow the isolation of interaction-negative mutants, the process of isolating the mutants is laborious because not all of the candidates obtained by screening carry the desired type of mutations (usually missense mutations). The most serious issue is that a significant fraction of candidates harbor frameshift or nonsense mutations, and it is necessary to perform western blotting to exclude undesired clones. To overcome this problem, new plasmid vectors have been developed4. In these vectors, KanMX, a drug resistance marker, is positioned out-of-frame downstream of the DB domain or AD. The marker gene becomes in-frame with the DB domain or AD only when the gene of interest is inserted. When a random mutation(s) is introduced in the gene of interest, undesirable mutants, such as those with frameshift or nonsense mutations, can be easily eliminated by performing drug resistance selection, and candidates carrying desirable missense mutations can be easily identified with the Y2H screen4. This article presents a protocol to isolate interaction-null/impaired mutants of a protein of interest using this strategy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA (8.0)</td>
			<td>Nacalai Tesque Inc.</td>
			<td>14347-21</td>
			<td></td>
		</tr>
		<tr>
			<td>10% SDS Solution</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>313-90275</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>135-07522</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>110-64785</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>021-07852</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Formedium</td>
			<td>AGR60</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>68-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HA-tag mAb-HRP-DirecT&#160;&#160;</td>
			<td>Medical &#38; Biological Laboratories Co. Ltd.</td>
			<td>M180-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA from salmon testes</td>
			<td>Merck KGaA.</td>
			<td>D1626</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck KGaA.</td>
			<td>9-0770-4-4L-J</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (Grade 50)</td>
			<td>Whatman, Cytiva</td>
			<td>1450-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (No.4A)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>01411090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for replicaplating (No.1)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>00011150</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418 Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>075-05962</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>230-37585</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>Nacalai Tesque Inc.</td>
			<td>20604-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>131-00405</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8226;12H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>10039-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31319-45</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;2H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31717-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td>AS ONE Corp.</td>
			<td>7-6200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>Nacalai Tesque Inc.</td>
			<td>25970-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid DNAs</td>
			<td>the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid isolation Kit</td>
			<td>Nippon Genetics Co. Ltd.</td>
			<td>FG-90502</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol #4,000</td>
			<td>Nacalai Tesque Inc.</td>
			<td>11574-15</td>
			<td></td>
		</tr>
		<tr>
			<td>SC double drop-out mix -Leu -Trp</td>
			<td>Formedium</td>
			<td>DSCK172</td>
			<td></td>
		</tr>
		<tr>
			<td>Seamless cloning kit (In-Fusion assembly )</td>
			<td>Takara Bio Inc.</td>
			<td>#639648</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>190-12865</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>3810-74-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymerase (GoTaq Green Master Mixes)</td>
			<td>Promega Corp.</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS (hydroxymethyl) aminomethane</td>
			<td>Formedium</td>
			<td>TRIS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai Tesque Inc.</td>
			<td>12967-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Nacalai Tesque Inc.</td>
			<td>35624-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (YNB)</td>
			<td>Formedium</td>
			<td>CYN0210</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>Nacalai Tesque Inc.</td>
			<td>07665-55</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating interaction-null&#47;impaired mutants using the yeast two-hybrid assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

We are interested in understanding the regulatory mechanisms of the cell cycle in Eukaryotes, especially the relationship between DNA replication, stable germ maintenance, and cell proliferation. The progression of the cell cycle is regulated by cyclin-dependent kinases, or CDKs, in Eukaryotes. This is the case for the initiation of DNA replication.We have identified a way, on substrates of CDKs, to initiate DNA replication. To understand the function of the protein of your interest, a comparison of the wild-type and mutant is a direct and effective way. Yeast two-hybrid assay, being simple detection and will enhance the process of isolating interaction-null mutants.This will contribute to advancement of research in the field.

Recently, it was found that the C-terminal half of Pol2 protein (Pol2-C) interacts with Mcm10. Both proteins are essential for the initiation of DNA replication and, hence, for cell growth in the budding yeast Saccharomyces cerevisiae13,14,15. To help understand the biological significance of this interaction, Pol2-C mutants that have no/diminished interaction with Mcm10 were isolated using the method described here.</p...

Watch this Scientific Journal Video about Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast at JoVE.com

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

isolating-interaction-nullimpaired-mutants-using-yeast-two-hybrid

Research

JoVE Journal

Biology

273 Views.  Kochi University of Technology. We are interested in understanding the regulatory mechanisms of the cell cycle in Eukaryotes, especially the relationship between DNA replication, stable germ maintenance, and cell proliferation. The progression of the cell cycle is regulated by cyclin-dependent kinases, or CDKs, in Eukaryotes. This is the case for the initiation of DNA replication.We have identified a way, on substrates of CDKs, to initiate DNA replication. To understand the function of the protein of your interest, a...